The neutrophil NADPH oxidase (NOX2), through the production of reactive oxygen species, is a key enzyme for host defense against invading pathogens. Although, it is established that the translocation of S100A8/S100A9, two Ca2+-binding proteins, is involved in NOX2 regulation, mechanisms underlining such a process remains elusive. Neutrophil-like HL-60 cells and human neutrophil were subjected to pharmacological inhibitors or transfected by specific siRNA. NOX2 activity and intracellular Ca2+ variations were quantified by spectrofluorimetry using Amplex red and Fura-2/AM for H2O2 and Ca2+ measurements. S100A8/A9 translocation was monitored by immunofluorescent labeling with Mac387 antibody. p38 MAPK activity was performed using a p38 MAPK activity test. Finally, protein-protein interactions were characterized by GST pull-down. Our data show that sphingosine kinases (SphK) are involved in the S100A8/A9 recruitment to the plasma membrane. Depletion of internal Ca2+ stores is required to mediate SphK activity dependent-S100A8/A9 translocation. Further, SphK knock-down resulted in a decrease of p38 MAPK activity. In addition, we observed that inhibition of S100A8/A9 translocation by SphK knock-down is associated to a decrease of NOX2 activation. S100A8/A9 interacts with cytosolic subunits of NOX2 (p67phox, p47phox, Rac1 and Rac2). These interactions are not inhibited by intracellular Ca2+ chelation. Ca2+ store depletion-induced SphK activity regulates NOX2 activity through the p38 MAPK dependent-translocation of S100A8/A9. Moreover, S100A8/A9 interacts with NOX2 cytosolic factors in a Ca2+ independent manner suggesting that S100A8/A9 regulates NOX2 assembly to the plasma membrane.